Gene expression in cells depends upon the sequential processes of transcription and translation. Together, these processes produce a protein from the nucleotide sequence of its corresponding gene.
Transcription involves the synthesis of mRNA from DNA by RNA polymerase. Transcription begins at a promoter region of the gene and continues until termination is induced, such as by the formation of a stem-loop structure in the nascent RNA or the binding of the rho gene product.
Protein is then produced from mRNA by the process of translation, occurring on the ribosome with the aid of tRNA, tRNA synthetases and various other protein and RNA species. Translation comprises three phases: initiation, elongation and termination. Translation is initiated by the formation of an initiation complex consisting of protein factors, mRNA, tRNA, cofactors and the ribosomal subunits that recognize signals on the mRNA that direct the translation machinery to begin translation on the mRNA.
Once the initiation complex is formed, growth of the polypeptide chain occurs by the repetitive addition of amino acids by the peptidyl transferase activity of the ribosome as well as tRNA and tRNA synthetases. The presence of one of the three termination codons (UAA, UAG, UGA) in the A site of the ribosome signals the polypeptide chain release factors (RFs) to bind and recognize the termination signal. Subsequently, the ester bond between the 3′ nucleotide of the tRNA located in the ribosome's P site and the nascent polypeptide chain is hydrolyzed. The completed polypeptide chain is released, and the ribosome subunits are recycled for another round of translation.
Mutations of the DNA sequence in which the number of bases is altered are categorized as insertion or deletion mutations (frameshift mutations) and can result in major disruptions of the genome. Mutations of the DNA that change one base into another are labeled missense mutations and are subdivided into the classes of transitions (one purine to another purine, or one pyrimidine to another pyrimidine) and transversions (a purine to a pyrimidine, or a pyrimidine to a purine).
Insertions, deletions, transition and transversion mutations can all result in a nonsense mutation, or chain termination mutation, in which the base mutation or frameshift mutation changes an amino acid codon into one of the three stop codons. The resulting premature stop codon can produce an aberrant, partially functional or non-functional protein in cells as a result of premature translation termination. A nonsense mutation in an essential gene can be lethal and can also result in a number of nonsense mutation mediated diseases.
In bacterial and eukaryotic organisms with nonsense mutations, a nonsense mutation can arise as a result of a germline or somatic mutation in DNA that results in the production of a premature termination codon. Readthrough of the premature termination codon allows insertion of a near-cognate tRNA at the ribosomal A-site, leading to synthesis of a full-length protein from an otherwise defective mRNA. Small molecules that affect cellular processes involved in protein synthesis and/or the mRNA quality control process (among other quality control processes) can promote readthrough at premature termination codons. Readthrough at a premature termination codon results when an amino acid is incorporated into the growing polypeptide chain at the site of the premature termination codon. The inserted amino acid may not necessarily be identical to the original amino acid of the wild-type protein; however, many amino acid substitutions are well tolerated and do not have a deleterious effect on protein structure or function. Thus, a protein produced by the suppression of the premature termination codon would be likely to possess activity close to that of the wild-type protein. Such a result provides an opportunity to treat diseases associated with nonsense mutations by avoiding premature translation termination through suppression of the premature termination codon.
Aminoglycosides have been suggested as possible candidates for premature termination codon suppression therapy. See Shalev and Baasov, Medchemcomm., 2014 August 1; 5(8): 1092-1105. However, to date, no structures of the human A-site are known to complex with any of the available aminoglycosides that induce readthrough. Moreover, elucidation of the readthrough mechanism of the aminoglycoside complex known as gentamicin in eukaryotic systems remains to be determined. Id. Since aminoglycoside activity in eukaryotes is not fully understood, the rational design of new and improved derivatives remains complex. Id. Therefore, there remains a need for further elucidation of the biological activity of aminoglycosides and the identification of new derivatives that avoid known toxicities while conferring the benefits of readthrough protein production.